ULTRAFAST TIME-RESOLVED CRYSTALLOGRAPHY ON SCAPHARCA DIMERIC AND TETRAMERIC H

鱼蚶二聚体和四聚体 H 的超快时间分辨晶体学

• 批准号: 8171975
• 负责人: WILLIAM E ROYER
• 金额: $ 2.56万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171975
• 关键词: Back; Binding; Biological Models; Clams; Computer Analysis; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Distal; Funding; Grant; Hemoglobin; Histidine; Human; Institution; Investigation; Kinetics; Ligands; Oxygen; Pathway interactions; Proteins; Research; Research Personnel; Resources; Rotation; Route; Signal Transduction; Solutions; Source; Structure; Time; United States National Institutes of Health; migration; mutant; research study; success; time use

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The clam, Scapharca inaequivalvis, possesses two hemoglobins that represent exceptional model systems for the investigation of protein allostery. Both hemoglobins bind oxygen cooperatively using a structural mechanism that is very different from the more well studied human hemoglobin. The dimeric hemoglobin, termed HbI, is the simplest possible model system for allostery with two identical subunits. Time-resolved crystallographic analysis of this hemoglobin provided, for the first time, a preliminary structural description of allosteric changes in real time (Knapp et. al. 2006, PNAS 103 7649-7654). Despite the overall success of these experiments, a major drawback was the very high level of geminate rebinding in the crystal, which substantially reduced the signal during the allosteric transition. Our analysis of ligand migration, including time-resolved crystallographic experiments, solution experiments and computational analysis (Knapp et al. 2009, Structure 17, in press) strongly suggests the crystal lattice restricts ligand exit by damping transient subunit rotations that are required for exit through a distal histidine gate. These experiments also revealed a potential alternate exit route through a "back door" channel. We are producing mutants that will allow ligands to exit through this back door within the tight confines of the crystal lattice. The tetrameric hemoglobin, termed HbII, is formed from two heterodimers, each of which has a similar assembly to that of HbI. The presence of two different subunits will permit investigation of how one subunit impacts a second subunit, which is not possible in the two-fold symmetric HbI. Therefore, we propose to use time-resolved x-ray diffraction experiments to elucidate the kinetic structural pathway in the tetrameric HbII and specific mutants of HbII.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 蛤蜊（Scapharca inaequivalvis）拥有两种血红蛋白，它们代表了研究蛋白质变构的特殊模型系统。 两种血红蛋白通过一种与经过深入研究的人类血红蛋白非常不同的结构机制协同结合氧。二聚血红蛋白，称为 HbI，是具有两个相同亚基的变构的最简单的模型系统。 这种血红蛋白的时间分辨晶体分析首次提供了实时变构变化的初步结构描述（Knapp 等人 2006，PNAS 103 7649-7654）。尽管这些实验总体上取得了成功，但一个主要缺点是晶体中成对重新结合的水平非常高，这大大减少了变构转变期间的信号。我们对配体迁移的分析，包括时间分辨晶体学实验、溶液实验和计算分析（Knapp 等人，2009 年，结构 17，出版中）强烈表明，晶格通过阻尼瞬态亚基旋转来限制配体退出，而瞬态亚基旋转是退出所需的。远端组氨酸门。这些实验还揭示了通过“后门”通道的潜在替代退出路线。我们正在生产突变体，允许配体在晶格的严格限制内通过这个后门退出。四聚体血红蛋白，称为 HbII，由两个异二聚体形成，每个异二聚体都具有与 HbI 相似的组装。两个不同亚基的存在将允许研究一个亚基如何影响第二个亚基，这在双重对称 HbI 中是不可能的。因此，我们建议使用时间分辨X射线衍射实验来阐明四聚体HbII和HbII特定突变体的动力学结构途径。